甜菜谷氨酰胺合成酶基因的克隆

【作者】 王淑春；

【导师】 康传红；

【作者基本信息】 黑龙江大学 ， 生物化学与分子生物学， 2007， 硕士

【摘要】 在植物的生长发育过程中，氮素的同化是一个十分重要的生理过程。其中，无机氮必须同化为有机氮才能为植物体所吸收和利用。谷氨酰胺合成酶是参与这一同化过程的关键酶。本实验的目的是从甜菜中克隆全长胞质型谷氨酰胺合成酶基因(GS1)。以甜菜叶片为材料，提取其基因组DNA为模板，根据GenBank上公布的甜菜GS1mRNA序列(索取号：AF343667)设计引物，进行PCR扩增。将PCR产物克隆到T载体中，转化大肠杆菌感受态细胞，测序。为检验实验结果的正确性，以甜菜叶片为材料，提取总RNA为模板，设计引物，进行RT-PCR扩增，把PCR产物克隆到载体，转化、测序。实验过程中对PCR体系进行了正交优化，并且使用了高保真酶，减少了PCR过程中碱基的错误掺入。由于GS1基因较大，于是采用了分段克隆的策略。实验中成功获得了甜菜全长GS1基因序列和部分GS1cDNA序列。全长的GS1基因长度为9606bp，包含13个外显子，被12个内含子分隔开，最大的内含子长3760bp，最小的为83bp。其外显子区与已公布的GS1mRNA序列的相似性达99.5％，只有5个碱基的差异。RT-PCR法获得了长为1068bp的GS1cDNA序列，与已知的GS1mRNA相似性达99.6％，只有4个碱基的差异。而两次实验中GS1基因外显子区与GS1cDNA的相似性达99.9％，只有1个碱基的差异。说明实验获得的数据准确可靠。本实验首次从甜菜中获得了全长GS1基因序列，可在此基础上进一步分离此基因的调控区，为此基因的表达调控研究及实现甜菜高同化氨途径奠定了分子生物学基础。更多还原

【Abstract】 Nitrogen assimilation is very important for growth and development in plants.Inorganic nitrogen must be assimilated as organic nitrogen which can be absorbed andused by plants. Glutarnine synthetase is a key enzyme during this process.The experiment is to acquire the whole cytosolic glutamine synthetase gene (GS1)from Beta vulgaris. DNA was extracted from sugar beet leaves as template, primerswere designed according to GS1mRNA sequence (accession no AF343667) fromGenBank. Then GS gene was amplified via PCR method. The PCR product was ligatedinto T vector, tansformed competent cell of E. coli and sequenced. In order to check theresult of PCR, total RNA was extracted from leaves as RT-PCR template, primers weredesigned as described above, then amplified by RT-PCR method. Cloning and sequencingof RT-PCR product was the same as that of PCR product.During the expetiment, PCR system was optimized by cross experiment, and highfidelity enzyme was utilized which can minimize wrong base integration. GS1 gene isvery long, so it was cloned by subsection PCR method. Full-length GS1 gene sequenceand partial GS1cDNA sequence of sugar beet was successfully obtained. The wholeGS1gene is 9606bp in length, including 13 exons which are separated by 12 introns.Among these introns, the longest one is 3760bp, and the shortest one is 83bp. Thesequence similarity between exon part of GS1 gene and known GS1mRNA is 99.5%,there are only 5 base difference, cDNA sequence is obtained by RT-PCR method whichis 1068bp in length, and the sequences similarity between this cDNA and knownGS1mRNA is 99.6%, there are 4 base difference. Meanwhile, the sequence similaritybetween exon part of GS1 gene and GS1cDNA in the experiment is 99.9%, there is onlyone base difference. We can see the results in this paper are pretty reliable.In this experiment, full-length GS1 gene was acquired firstly from sugar beet. Thenthe regulatory region can be separated based on the result, which provides molecularbiological foundation for GS gene expression, regulation and implement of highammonia assimilation in sugar beet更多还原